To date, some culturing methods for inducing differentiation of nerves from a pluripotent stem cell such as an ES cell have been known, including those reported by the present inventors (Non-Patent Documents 1-3, Patent Documents 1 and 2); there are high expectations for ES cell-derived nerve cells (e.g., dopamine nerve cells and the like) as a source of graft cells for cell transplantation therapy in regenerative medicine for intractable neurologic diseases. To this end, disease-related nerve cells that are present in the brain must be produced accurately; however, because an extremely large number of kinds of nerve cells are present in the brain, there are still many types of nerve cells and brain tissues for which efficient in vitro differentiation has been unsuccessful.
The cerebrum, particularly cerebral cortex, is the center of higher brain functions; a disorder thereof causes serious motor, mental, and cognitive disorders. For example, Alzheimer's disease, cerebral infarction, epilepsy, motor neuron disease (ALS) and the like can be mentioned. For the treatment of cerebral disorders, etiologic research, drug discovery research, cell transplantation therapy research and the like have been conducted so far, but it is extremely difficult to obtain human cerebral tissue for the sake of these researches. Although it has recently become possible to induce differentiation of an embryonic stem cell into cortical progenitor cells (see Non-Patent Document 4), it has been difficult to control the selective induction of differentiation from those progenitor cells to particular cerebral cortical neurons.
The present inventors showed that dispersion suspension culture using a serum-free medium (the SFEB method) is effective as a method of inducing differentiation of a pluripotent stem cell such as animal or human ES cell into nerves (see Non-Patent Documents 3 and 4 and Patent Document 1). This method enables efficient induction of differentiation into nerve cells and sensory cells of the forebrain, particularly of the cerebrum and the neural retina. The present inventors also succeeded in inducing differentiation into brainstem tissues such as the cerebellum by adding growth factors such as Wnt to the medium.
However, an analytical study with mouse embryonic stem cells revealed that when the SFEB method was applied, about 30% of the cells differentiated into cerebral nerve cells, but the remaining majority occurred as a mixture of other kinds of nerve cells. Additionally, cerebral cortex cells accounted for only about 40% of the differentiation-induced cerebral nerve cells; the induction efficiency was not so high. Furthermore, the majority of the cerebral tissues induced by a conventional method such as the SFEB method failed to have a clear morphology of cortical tissue, mostly forming disarrayed cell masses. Additionally, the conventional SFEB method does not enable efficient induction of differentiation of the tissue of the rostral diencephalon, which develops on the most rostral side of the central nervous system, particularly of the hypothalamus, from an ES cell.